For a polypeptide comprising a plurality of nuclease subunits consisting of a DNA-binding domain and a function domain, TALEN (TALE Nuclease), ZFN (Zinc Finger Nuclease) and the like are known (Patent references 1-4, Non-patent references 1-5). For instance, an artificial nuclease is known in which a DNA-cleaving domain is used as a function domain. These artificial nucleases cause cleavage of DNA duplicates by a multimer formed by a plurality of DNA-cleaving domains approaching close together around a binding site of a DNA-binding domain. A DNA-binding domain comprises repetition of plurality of DNA-binding modules and the respective DNA-binding module recognizes a specific base pair in a DNA strand. Thus, by suitably designing DNA-binding modules, it becomes possible to specifically cleave a sequence of interest. By utilizing errors and recombinations that occur when sequence specific cleavage is subject to repair, it is possible to introduce deletion, insertion and mutation of a gene on a genomic DNA. Therefore, these nucleases may be applied for various genetic modifications such as a genome editing (cf. Non-patent reference 6).
Non-patent reference 1 discloses a polypeptide in which a DNA-binding domain and a function domain are linked to each other via a linker of 47 amino acid residues. Non-patent reference 1 discloses a nuclease in which a single amino acid residue in a specific site in a DNA-binding module other than a DNA recognition site is periodically varied for the every four DNA-binding modules used. However, Non-patent reference 1 fails to disclose that the used polypeptide has compatibility between a high level of desired function of a function domain and a high level of specificity of sequence recognition of a DNA binding domain. Non-patent reference 1 also fails to disclose that a plurality of amino acid resides in a DNA-binding module are periodically varied. Non-patent reference 1 also fails to disclose the significance and the purpose of the periodical variation.
Non-patent reference 2 discloses a polypeptide in which a DNA-binding domain and a function domain are linked to each other via a linker of 63 amino acid residues. Non-patent reference 2 also discloses a polypeptide in which amino acid residue(s) in a DNA-binding module other than those amino acid residues in a DNA recognition site is/are periodically varied for the every four DNA-binding modules used. However, Non-patent reference 2 fails to disclose that the used polypeptide has compatibility between a high level of desired function of a function domain and a high level of specificity of sequence recognition of a DNA-binding domain. Non-patent reference 2 also does not refer to anything about the purpose and the significance of the periodical variation for every four DNA-binding modules.
Non-patent reference 3 discloses a polypeptide in which a DNA-binding domain and a function domain are linked to each other via a linker of 47 amino acid residues. Non-patent reference 3 also discloses a polypeptide in which amino acid residue(s) in a DNA-binding module other than those amino acid residues in a DNA recognition site is/are varied at random for the every DNA-binding modules used. However, Non-patent reference 3 fails to disclose that the used polypeptide has compatibility between a high level of desired function of a function domain and a high level of specificity of sequence recognition of a DNA-binding domain. Non-patent reference 3 also fails to disclose the purpose and the significance of the variation at random. Non-patent reference 3 also fails to disclose the periodical variation for every DNA-binding module.
Non-patent reference 4 discloses a polypeptide in which a DNA-binding domain and a function domain are linked to each other via a linker of 63 amino acid residues. Non-patent reference 5 discloses a polypeptide in which a DNA-binding domain and a function domain are linked to each other via a linker of 47 amino acid residues. However, neither Non-patent reference 4 nor Non-patent reference 5 discloses that the used polypeptide has compatibility between a high level of desired function of a function domain and a high level of specificity of sequence recognition of a DNA-binding domain. Also, neither Non-patent reference 4 nor Non-patent reference 5 discloses the periodical variation for every DNA-binding module.